L11-binding fragment of 23S rRNA folds into a specific tertiary structure. This structure is stabilized by Mg2+ and some other divalent metal ions. The presence of divalent metal ions is necessary for the formation of this tertiary structure in 100 mM monovalent salt solutions. The tertiary structure is also stabilized by NH4+ in preference to other monovalent cations. The tertiary structure can be observed in 1.6 M NH4Cl even in the absence of divalent metal ions, however millimolar concentrations of those ions render it considerably more stable. GACG sequence variant of the L11-binding fragment of 23S rRNA provides a particularly convenient model for investigation of specific interactions of divalent metal ions with the tertiary structure, as the tertiary structure unfolding transition is well separated from the higher temperature transitions of secondary structure unfolding. The tertiary structure unfolding is seen as a peak on UV melting profiles. As more divalent metal ion (such as Mg2+) is added to the RNA solution, this peak moves to higher temperatures. It also sharpens, which indicates that either there is a considerable Dcp associated with tertiary structure unfolding, or there is a considerable enthalpy (on the order of 10 kcal/mole) associated with Mg2+ ion binding. The ITC experiment performed at the biocalorimetry center allowed us to rule out the latter. No measurable heat effect of titrating Mg2+ into RNA solution in the presence of 1.6 M NH4Cl was observed, which put an upper limit of ~0.25 kcal/mole for Mg2+ binding to this RNA.